Severe acute respiratory syndrome (SARS) was first identified in late November 2002 in Guangdong Province, China. In the ensuing months, major outbreaks were reported in other parts of China, Vietnam, Canada, Singapore, Taiwan, and elsewhere in the world. The disease is unusual in its high level of infectivity, as demonstrated among the health care workers and family members that have been in close contact with infected individuals. In addition, it has also been reported that infected patients do not respond to empirical antimicrobial treatment for acute community-acquired typical or atypical pneumonia (Peiris et al. (2003) “Coronavirus as a possible cause of severe acute respiratory syndrome,” Lancet 361:1319-1325, which is incorporated by reference).
The cause of SARS has been identified as a novel coronavirus (CoV) (Drosten et al. (2003) “Identification of a novel coronavirus in patients with severe acute respiratory syndrome,” N. Engl. J. Med. 348:1967-1976, which is incorporated by reference), because clinical specimens from patients infected with SARS revealed the presence of crownshaped CoV particles. This new CoV has thus been referred to as SARS CoV. The full-length genome sequence of the SARS CoV has been reported from different isolates, and the genome organization of SARS CoV was found to be similar to that of other CoVs (Marra et al. (2003) “The genome sequence of the SARS-associated coronavirus,” Science 300:1399-1404 and Ruan et al. (2003) “Comparative full-length genome sequence analysis of 14 SARS coronavirus isolates and common mutations associated with putative origins of infection,” Lancet 361:1779-1785, which are both incorporated by reference).
CoVs are a family of positive-strand RNA-enveloped viruses called Coronaviridae, which are now categorized under the newly established order Nidovirales. This order comprises the families Coronaviridae and Arteriviridae. The name Nidovirales comes from the Latin word nidus, for nest, referring to the 3′-coterminal “nested” set of subgenomic mRNAs produced during viral infection (Cavanagh (2003) “Nidovirales: a new order comprising Coronaviridae and Arteriviridae,” Arch. Virol. 14:629-633, which is incorporated by reference). The SARS CoV genome is very large, 29.7 kb (Marra et al. (2003), supra, and Ruan et al. (2003), supra, which are both incorporated by reference), and encodes 23 putative proteins. Major structural proteins include nucleocapsid, spike, membrane, and small envelope. Nonstructural proteins include the papainlike proteinase, 3C-like proteinase, RNA-dependent RNA polymerase (RdRp), helicase, and many other proteins involved with viral replication and transcription (Cavanagh (2003), supra, and Ng et al. (2002) “Membrane association and dimerization of a cysteine-rich, 16-kilodalton polypeptide released from the C-terminal region of the coronavirus infectious bronchitis virus 1a polyprotein,” J. Virol. 76:6257-6267, which are both incorporated by reference). In other CoVs, many of the nonstructural proteins are only slightly conserved in the viral sequence, the exception being RdRp, which is highly conserved in many CoVs.
The use of oligonucleotide sequences as primers and/or probes for the recognition of infectious agents is one alternative to problematic immunological identification assays and other pre-existing methodologies. For example, nucleic acid probes complementary to targeted nucleic acid sequences have been used in hybridization procedures, such as Southern blots and dot blots, to detect the target nucleic acid sequence. Many of these hybridization procedures have depended on the cultivation and/or enrichment of the organism and, thus, are generally unsuitable for rapid diagnosis. The advent of techniques for the rapid amplification of specific nucleic acid sequences, such as the polymerase chain reaction among many others, has provided a mechanism to use primer and probe nucleic acids directly on clinical specimens, thereby eliminating enrichment and in vitro culturing of the pathogen prior to performing the hybridization assay. Thus, amplification-based hybridization assays can provide simple and rapid diagnostic techniques for the detection of pathogens, such as the SARS CoV in clinical samples.